This invention relates to the efficient collection and detection of low levels of radiation arising within microliter volumes of sample.
The radiation may be in the form of luminescence from a chemical reaction or result from the interaction of an intense light source and the sample. Alternatively, the processes of light scattering, Raman scattering, fluorescence and phosphorescence may be used.
The technique of light scattering is particularly useful in the detection of particles, and two broad classifications of instruments can be identified. Particles of diameters significantly smaller than the wavelength of probe light scatter isotropically, i.e. equally in all directions. This type of scattering is usually called Rayleigh scattering. Particles of diameters significantly larger than the wavelength of probe light scatter primarily in the forward direction and, often, the intensity of scatter is a complicated function of scattering angle. This type of scattering is usually called Mie scattering. The intensity of forward scattered light varies as the sixth power of the particle diameter; hence, special precautions must be taken when attempting to measure the light scattered from very small particles.
The present invention permits for the determination of relative particle size and/or concentration of very small particles, particularly in a flowing system.
Intense light sources such as lasers are used for studying small particles. The use of such sources introduces problems associated with discrimination of light scattered by instrumental components from that scattered by the sample. The intensity of light scattered from small particles may be less than one millionth the intensity of laser radiation. To measure such low intensities, it is necessary to collect as much of the light scattered by the sample as possible, yet reject that scattered by the instrument. In general, it becomes increasingly difficult to eliminate instrumental light scattering as the angle of detected scattering decreases. Consequently, when measuring the light scattered by particles significantly smaller than the wavelength of probe light, it is best to avoid detection of the small angle scattering, while collecting as large a solid angle of scattered light as possible.
When measuring the light scattered by small particles, considerable attention must be paid to the overwhelming level of light scattered from unwanted foreign particles of large size. For this reason, it is advantageous to keep the size of the scattering volume (volume of sample simultaneously illuminated and detected) as small as possible. In a flowing sample, these particles will be evidenced as signal "spikes" abruptly rising from a base signal. The base signal represents the signal from the more numerous small particles.
Large angle scattered light instruments described in the prior art have used large scattering volumes and require extensive sample treatment to minimize the detection of large foreign particles. Furthermore, sample volume has to be large to avoid detection of light scattered from the instrument. These prior art instruments have usually used lenses and apertures to define the scattering angle and, of necessity, capture a small solid angle of scattered light.
Moreover, most of the prior art has addressed the detection of relative large biological cells (cytometry). The signals from these large particles are much greater than those provided by the small particles of particular interest in the present invention.
Raman scattering is used for the chemical characterization of the samples rather than particle size. Like Rayleigh scattering, the signal intensities are very low and it is important to capture as large a solid angle of Raman scattered light as possible while also using very intense sources of incident radiation. The Raman scattered signals are of different wavelength from the incident light. Chemical characterization of the sample is determined by the difference between the frequency of the Raman signal and the frequency of the exciting radiation. It is customary to use a blocking filter to reject the incident radiation followed by a monochromator to determine the frequency of the Raman signal. While this frequency difference helps in the discrimination between exciting and Raman signals, the Raman bands are often narrow; hence, the signals are very weak. It is important to use whatever geometric means are available to minimize the detection of the incident radiation and maximize the detection of the weak Raman radiation from the sample.
Fluorescence techniques are used when the sample or some component(s) of the sample can be tagged with a fluorescing dye. The sample is then excited by an intense beam of exciting radiation, usually from a laser. The wavelength of the exciting radiation is chosen to correspond to the wavelength of maximum sample excitation. The wavelength of the fluorescing radiation will usually be greater than the exciting wavelength, and a filter or monochromator will be chosen to pass only that radiation corresponding to the wavelength of maximum fluorescence emission. When the sample concentration is low, great care is needed in the choice of filters or monochromators in order to discriminate between exciting and fluorescing wavelengths. No filters provide a perfect discrimination, and it is desirable to use whatever geometric means are available to minimize the detection of exciting rays scattering from the instrument. Under ideal conditions, tagged molecules can be detected at extremely low concentration.
Phosphorescence techniques are employed where the sample, itself, or the tagged sample continues to emit radiation a significant time after excitation. Time as well as wavelength can then be used to discriminate between exciting and emitting radiation.
Luminescence techniques are used when the sample or some component of the sample emits radiation as a result of a chemical reaction of the sample with a reagent. No external exciting radiation is needed; hence, all of the radiated light may be detected without recourse to filtering. However, the luminescence signals are usually very weak and depend on the number of reacting molecules within the detected sample volume. A compromise must usually be made between detected sample volume and concentration of reacting molecules. The efficient collection and detection of the radiation emitted within the detected sample volume is of great importance.
There is a need to provide a system for radiation collection and detection which provides significant advances over the prior art.